The present invention relates to novel viral sequences and various uses thereof. In particular it is based on identification of a hitherto unrecognised sequence at the 3xe2x80x2 end of the GBV genome. Nucleic acid with the sequence, or allelic variants and fragments thereof, are useful in providing viral vector constructs infective in tamarins. Hybrid viral vectors including HCV components can be provided including nucleic acid of the invention allowing for infection of tamarins as a suitable model for study of HCV and for testing agents for ability to inhibit HCV activity.
The hepatitis GB agent was first described by Deinhardt and co-workers (Deinhardt et al. 1967. J. Exp. Med), who inoculated tamarins, small primates of the Saguinus species, with a serum from a patient (whose initials were GB) affected by acute hepatitis. This serum induced hepatitis in all inoculated tamarins and was passaged serially in these animals. Only recently molecular characterization of this agent has been achieved (Simons et al. 1995. Proc. Natl. Acad. Sci.; Muerhoff et al. 1995. J. Virol.), showing that two distinct positive-strand RNA viruses, GB virus A and B (GBV-A, GBV-B), were associated with GB agent hepatitis.
Subsequently, it was shown that the two viruses could be passaged separately in tamarins and that GBV-A does not replicate in the liver whereas GBV-B causes hepatitis (Schlauder et al. 1995. J. Med. Virol.; Schlauder et al. 1995. The Lancet). The initial inoculum originates from humans, although formal proof that GBV-A and GBV-B are indeed human pathogens has yet to be obtained. Immune reactivity against GBV-A and GBV-B derived peptides has been found in humans (Pilot-Matias et al. 1996. J. Med. Virol.; Simons et al. 1995. Nature Medicine), but attempts to detect viral sequences in the reactive plasma have to date been unsuccessful (Simons et al. 1995. Nature Medicine).
Sequence analysis of GBV-A and GBV-B genomes suggested that they belong to the Flaviviridae family and are related to hepatitis C virus (HCV). Comparison of their open reading frame with that of different HCV strains shows from 26 to 33% identity at the amino acid sequence level. However, comparison of the hydropathy profiles suggests that, despite the little overall amino acid sequence identity, these viruses share similarity in protein structure (Muerhoff et al. 1995. J. Viral.; Ohba et al. 1996. FEBS Letters).
Of the two, the most closely related to HCV is GBV-B, not only for the sequence similarity but also for sharing functional characteristics, tissue tropism, since they are both hepatotropic viruses, and pathogenesis (Zuckerman et al. 1995. The Lancet; Simons et al. 1996. Viral Hepatitis Reviews). The hypothesis of a functional homology between proteins of the two viruses has been already validated, in the case of proteins with important enzymatic activity such as the NS3 serine protease, responsible of maturation of the viral polyprotein (Scarselli et al. 1997. J. Virology) and the RNA dependent RNA polymerase NS5B protein responsible for the replication of the viral genome (unpublished data).
GBV-B may be used as a model for HCV with regard to infection and pathogenesis. In particular, HCV/GBV-B recombinant viruses may be used to evaluate the effect of antivirals directed against HCV targets of interest in vivo. This kind of approach requires the availability of a GBV-B genomic molecule able to replicate in vivo. The art has so far failed to provide this.
Only one GBV-B genomic sequence has been reported (GenBank Accession No. U22304) spanning 9143 nucleotides. The ORF of GBV-B shows a significant homology to the corresponding regions of HCV, and the potential secondary structure of GBV-B 5xe2x80x2-UTR shows a striking similarity to that of HCV (Lemon and Honda. 1997. Seminars in Virology). To date there are no reports of replication in vivo of any RNA based on this sequence.
The present invention is based at least in part on the identification of a novel nucleotide sequence, called GBV-B 3xe2x80x2X, that the inventors have found to be an integral part of the genome of GB virus B (GBV-B), corresponding to the 3xe2x80x2-terminus of the GBV-B genome.
In natural isolates this sequence is present: on both strands of GBV-B RNA, it was found in GBV-B RNA extracted from both serum and liver of tamarins and in GBV-B RNA extracted from at least two species of tamarins.
The putative secondary structure of this novel sequence resembles (particularly the 3xe2x80x2-terminal stem-loop structure) that of HCV, whose corresponding sequence has been proved to be essential for replication and infection (Kolykhalov et al. 1997. Science, 277: 570-574; Yanagi et al. 1999. Proc. Natl. Acad. Sci. USA, 96: 2291-2295).
The invention allows for this sequence to be used to confer infectivity in Tamarins.
Recombinant HCV/GBV-B viruses may be constructed carrying the HCV target genes of interest, i.e. for example a sequence coding for the NS3 protease, the NS5B polymerase, another single HCV protein (selected from E1, E2, NS2, NS4A, NS4B, and NS5A) or a combination of any of these, that are infective in tamarins, for example as described below.
A hybrid GBV/HCV comprising HCV NS3/4a represents one preferred embodiment of the invention. NS3/4a has been shown to cut the GBV sequence in the correct places. Other preferred embodiments comprise HCV helicase and/or polymerase.
At present, the only animal model for HCV infection is the chimpanzee. The limitations that this animal model implies, the chimpanzee being a protected species, make it inconvenient to use it in general and impossible for pharmacological studies in particular, where a large number of animals of small size is required. So far only a very limited number of chimpanzees per experiment has been experimentally infected with HCV to test infectivity of inocula and in trials for vaccine development.
The present invention makes it possible to infect small sized primates (tamarins) with recombinant GBV-B infectious RNA carrying HCV sequences encoding for HCV pharmacological targets. This means that it is possible to accomplish studies on anti-HCV antivirals in more suitable primates, thus overcoming the limitations in the number of animals to be used and dramatically reducing the cost of the experiments.